A subset of G-protein coupled receptors are highly effective mediators of cell proliferation, gene expression and cytoskeletal rearrangement. What characterizes these receptors is their ability to strongly couple, through G 12/13 proteins, to activation of the low molecular weight protein RhoA. The ligands for these receptors include the lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid, as well as thrombin, thromboxane A-2 and various chemokines. A unifying concept is that these GPCR agonists are inflammatory mediators, formed or released from cells that are activated in response to stress or injury. Effects of these ligands on cells can be either protective or deleterious depending on the cell type involved and the acute vs chronic nature of the stimulation. We hypothesize that the acute effects of these GPCRs, through activation of RhoA and concomitant activation of Akt, subserve a protective function in cardiomocytes. Studies proposed in Aim #1 will examine protective effects of S1P in ischemia reperfusion in vivo, in isolated perfused hearts and in cardiomyocytes. Protective effects will be attributed to activation of specific S1P receptor subtypes using S1P receptor knockout (KO)mice. The involvement of RhoA,Akt, eNOS and NFKB as downstream protective mediators will be assessed in cardiomyocytes from S1P receptor and KO mice, and by studies using inhibitors of these signaling pathways. Aim #2 defines mechanisms by which RhoA mediates cardioprotection and affects cell survival. Proposed studies will determine whether RhoA activation or overexpression in cardiomyocytes induces NFKB and consequent changes in its downstream early gene targets including lAPs, TNFa and 1L-6 family cytokines. Transgenic mice with inducible cardiac RhoA expression will be used to directly relate changes in RhoA activity to cardioprotection or apopotosis and explore the underlying mechanisms. Studies proposed in Aim #3 examine the hypothesis that Akt causes acute cardioprotection via effects on mitochondrial function. The ability of agonists and interventions that activate Akt to induce cardioprotection is investigated, focusing on Akt effects on the mitochondrial permeability transition (PT)pore and the ability of Akt to interact directly with components of the PT pore. Biochemical and single cell studies using fluorescent indicators and FRET based Akt reporters are proposed. The possibility that Akt interacts with and phosphorylates hexokinase II (HK-II) to regulate PT pore activity is specifically examined. Understanding the survival pathways actived by S1P through RhoA and Akt could provide a means of arresting myocyte death resulting from ischemic injury, and the subsequent initiation of inflammation and remodeling. Early intervention specifically targeted at these cardiomyocyte protective pathways could therefore be useful in preventing development of heart failure.